Non-coding RNAs (ncRNAs) are not translated into protein but are functional in regulation of transcription. There is strong evidence that ncRNAs are key players in developmental, cellular, and immunological processes. One class of ncRNAs are microRNAs (miRNAs), discovered in 1993, that are believed to regulate almost 30% of all human genes. Each miRNA has a few to as many as 200 target genes, which they modulate by inhibiting mRNA at a transcriptional level (mRNA degradation) or the translational level (resulting in inhibition of protein synthesis). There are now over 670 known human miRNAs (Sanger miRBase) though only a few are mechanistically validated. Current literature identifies 30 miRNAs associated with hematopoietic lineage cells but only a small subset documented in T and B cells. We hypothesize that miRNAs play a pivotal role in regulation of human immunity by targeting key differentially and constitutively expressed genes regulating T and B lymphocyte activation, differentiation and survival. Despite many studies describing immune-associated transcripts and proteins orchestrating the transplant immune response, little is known about regulating this genomic complexity. The premise of this proposal is that identifying the miRNA- regulated molecular networks within this complexity will illuminate the key pathways in a way that cannot be achieved by simple gene expression profiling or proteomics. To test this premise in our preliminary studies we used Whole Exon Arrays for mRNA and a novel stem-loop qPCR method for 420 known miRNAs to study human T and B cell activation. Results revealed 62 differentially expressed miRNAs of which 42 have not previously been linked to lymphocyte or hematopoietic lineage regulation, over 3,000 differentially expressed mRNAs, unique miRNA profiles distinguishing T and B cell activation and a number of high value candidates for miRNA regulation of biologically significant molecular pathways. The objective of this proposal is to complete a full analysis of miRNA expression in human T and B cell activation, to investigate the novel possibility that post-transcriptional processing of precursors for miRNA in the nucleus is important, to use tandem mass spectrometry proteomics and Next Generation deep sequencing (Roche 454 FLX) to discover the correlations between protein, mRNA and miRNA expression and molecular network regulation during activation, and to validate a number of high value miRNA candidates in biologically significant pathways. If our multidimensional omics approach is successful, then it is a proof of concept for an effort to organize the full force of a large collaborative group at validating all the major pathways for each cell type, identifying the impact of immunosuppression, correlate results with clinical samples from transplant patients and aim to advance our understanding of transplant immunology from the level of individual gene-linked mechanisms to integrated molecular networks.